이 작품은 중국 국립 자연과학 재단(31501099호)과 중국 후베이성 교육부중장기 및 영(No. 20191104년). 그리고, 우리는 마우스 배아 줄기 세포주 A2lox를 제공하는 우한 과학 기술 대학의 웬성 덩 교수에게 감사드립니다.

1. 마우스 배아 줄기 세포 배양
<ol><li>0.1% 젤라틴 코팅 세포 배양 접시 또는 접시를 준비하십시오.<ol>
<li>멸균 된 0.1 % 젤라틴 (물에서 0.1 % w / v)의 2 mL을 60mm 세포 배양 접시에 추가하십시오. 셀 배양 접시의 코팅도 보장하기 위해 부드럽게 흔들어.</li>
		<li>37°C에서 5% CO2 인큐베이터에 접시를 넣고 1시간 동안 코팅할 수 있습니다.</li>
		<li>세포를 파종하기 전에 0.1% 젤라틴 용액을 제거합니다. 참고: 젤라틴을 제거한 후에는 코팅된 접시를 말리거나 씻을 필요가 없습니다.</li>
	</ol>
</li>
	<li>마우스 배아 줄기 세포 (A2lox 및 129) 배양<ol>
<li>인큐베이트 mESC(A2lox 및 129) 세포에서 0.1% 젤라틴 코팅 60 mm 세포 배양 식 에서 mESC 성장 배지에서 37°C에서 각각 5%CO2 인큐베이터에. mESC 성장 매체는 85% 노크 아웃 DMEM/F12, 15% 녹아웃 세럼 교체(KSR), 0.1mM β-메르카토에탄올(2ME), 2mM 글루타맥스, 1% 비필수 아미노산 (NEAA), 1 % 페니실린 / 연쇄 상피신 (P / S), 1000 U / mL 백혈병 억제 인자 (LIF), 10 nM CHIR-99021 (GSK-3 억제제) 및 0.33 nM PD032590 (MEK 억제제). 주의: β-메르카토에탄올은 인화성이며 흡입 독성이 있습니다. 화재 원인에서 멀리 하고 사용 시 흡입을 피하기 위해 마스크를 착용하십시오.</li>
		<li>A2lox와 129의 더 나은 성장을 위해 매일 mESC 성장 매체를 변경합니다.</li>
		<li>세포가 80%에 도달하면 배지를 제거하고 접시에 0.1% 트립신1mL을 추가합니다. 모든 셀에 트립신의 커버를 보장하기 위해 부드럽게 30 s에 대한 바위.</li>
		<li>약 1 분 동안 세포를 두고 트립신이활성화한 다음 1 mL 파이펫을 사용하여 트립신을 제거하십시오.</li>
		<li>접시에 mESC 성장 배지 2mL을 추가하고, 파이펫을 여러 번 위아래로 사용하여 단일 셀 서스펜션을 만듭니다.</li>
		<li>혈종계를 사용하여 현탁액내 의 세포 밀도를 가능한 한 정확하게 계산합니다.</li>
		<li>세포를 7그룹으로 나누고 표 1에표시된 다른 프로토콜을 사용하여 분화를 유도한다.</li>
	</ol>
</li>
</ol>2. MESC에서 NPC로의 차별화 (1단계)
<ol><li>0.1% 젤라틴 코팅 세포 배양 플레이트 또는 커버립을 준비합니다.<ol>
<li>사용하기 전에 1.1 단계에서와 같이 0.1 % 젤라틴 코팅 6 웰 플레이트 또는 커버립을 준비하십시오.</li>
	</ol></li>
	<li>프로토콜 1을 사용하여 단계 I 분화(표 1)<ol>
<li>약 2 x 104 mESC를 기초 분화 배지 의 2 mL에 씨를 뿌리고 0.1 % 젤라틴 코팅 6 웰 플레이트에서 잘 합니다. 현미경의 밑에 세포 조밀도를 확인하십시오.</li>
		<li>부착을 허용하기 위해 6h에 대해 5% CO2 인큐베이터에서 37°C에서 세포를 배양한다.</li>
		<li>부착 후 인큐베이터에서 세포를 꺼내 PBS 2 mL로 두 번 씻어 내십시오.</li>
		<li>기초 분화 배지I(표 1)의2mL을 각 웰에 넣고 세포를 인큐베이터에 다시 넣습니다.</li>
		<li>8 일 동안 분화를 위해 세포를 둡니다. 2일마다 기저 분분 매체 I를 교체하십시오.</li>
	</ol></li>
	<li>프로토콜 2를 사용하여 단계 I 분화(표 1)<ol>
<li>1.5 x 106 mESC를 기초 분화 배지 10mL에 첨가하여 5% CO2 인큐베이터에서 37°C에서 배아 체형성을 허용합니다.</li>
		<li>2 일 후, 15 mL 원심 분리 튜브로 세포 응고를 전송하고 중력에 의해 정착할 수 있습니다.</li>
		<li>상체를 제거하고 신선한 기초 분화 배지 10mL를 추가하여 배아 체를 재보습합니다. 새로운 미각 세균 성 접시에 그들을 심고 또 다른 2 일 동안 차별화를 허용.</li>
		<li>현미경(도 2A)하에서배아체의 형성을 확인한다.</li>
		<li>단계 2.3.2-2.3.3에 설명된 바와 같이 배아 체를 수집한다. 약 50개의 배아체를 기초 분화 배지의 2mL로 약 50개의 배아체를 잘 하여 0.1% 젤라틴 코팅 6웰 플레이트에 시드한다.</li>
		<li>1mM 올트랜스 RA 스톡(DMSO)을 준비하고 서브 패키징 후 -80°C 냉동고에 빛으로부터 멀리 보관하십시오. 참고: RA는 불안정하며 RA 재고를 준비하는 동안 빛을 멀리하고 공기 접촉을 줄이는 데 주의를 기울여야 합니다.</li>
		<li>RA 유도의 경우, RA 스톡 2μL을 각 웰에 추가하여 1 μM의 최종 농도를 만듭니다.</li>
		<li>플레이트를 37°C에서 5%CO2 인큐베이터에 넣고 또 다른 4일 동안 분화한다.</li>
		<li>2일마다 기초 분화 매체 I(1 μM RA)의 전체 2mL를 변경합니다.</li>
	</ol></li>
	<li>프로토콜 3을 사용하여 단계 I 분화(표 1)<ol>
<li>식물 1.5 x 106 mESC는 10mL의 기초 분화 배지에서 미적 세균 성 접시에 들어가면 5 % CO2 인큐베이터에서 37 °C에서 배아 체 형성을 위해 2 일 동안 둡니다.</li>
		<li>현미경(도 2B)하에서배아체의 형성을 확인한다.</li>
		<li>세포 응집체를 15mL 원심분리기 튜브로 옮기고 중력에 의해 정착하게 합니다.</li>
		<li>상체를 조심스럽게 제거하고 신선한 기초 분화 매체 10 mL을 추가하여 다시 일시 중단합니다.</li>
		<li>약 50개의 배아체를 기초 분화 배지 의 2mL로 약 50개의 배아체를 잘 하여 0.1% 젤라틴 코팅 6웰 플레이트에 시드한다.</li>
		<li>RA 유도의 경우, RA 스톡 2μL을 각 웰에 추가하여 1 μM의 최종 농도를 만듭니다.</li>
		<li>플레이트를 37°C에서 5%CO2 인큐베이터에 넣고 6일 동안 분화한다. 전체 기저 분분 매체 I(1 μM RA)를 2일마다 변경합니다.</li>
	</ol></li>
	<li>프로토콜 4를 사용하여 단계 I 분화(표 1)<ol>
<li>약 2 x 104 mESC를 기초 분화 배지 의 2 mL 내에서 종자 0.1% 젤라틴 코팅 6 웰 플레이트에 잘. 플레이트를 37°C에서 5% CO2 인큐베이터에 넣고 6h에 부착할 수 있도록 합니다.</li>
		<li>부착 후 PBS 2mL로 셀을 두 번 씻으시면 됩니다. 각 웰에 2mL의 기초 분화 배지 I를 추가하고 37°C에서 5% CO2 인큐베이터에서 4일 동안 분화를 허용한다.</li>
		<li>RA 유도의 경우, 각 우물(작동 농도는 1 μM)에 2 μL의 모든 트랜스 RA 스톡을 추가하여 또 다른 4일 동안 분화를 유도합니다.</li>
		<li>전체 공정에서 2일마다 전체 매체를 교체하십시오.</li>
	</ol></li>
	<li>프로토콜 5를 사용하여 단계 I 분화(표 1)<ol>
<li>약 2 x 104 mESC를 기초 분화 배지 의 2 mL 내에서 종자 0.1% 젤라틴 코팅 6 웰 플레이트에 잘. 플레이트를 37°C에서 5% CO2 인큐베이터에 넣고 6h에 부착할 수 있도록 합니다.</li>
		<li>PBS의 2mL로 세포를 두 번 씻으시면 됩니다. 각 웰에 2mL의 기초 분화 배지 I를 추가하고 37°C에서 5% CO2 인큐베이터에서 2일 동안 분화를 허용한다.</li>
		<li>후속 RA 유도의 경우, 각 우물에 2 μL의 RA 스톡을 추가하여 1 μM의 최종 농도를 만듭니다. 플레이트를 37°C에서 5% CO2 인큐베이터에 배치하여 또 다른 6일 동안 분화를 유도한다.</li>
		<li>전체 공정에서 2일마다 전체 매체를 교체하십시오.</li>
	</ol></li>
	<li>프로토콜 6을 사용하여 단계 I 분화(표 1)<ol>
<li>식물 1.5 x 106 mESC는 배아 체 형성을 허용하기 위해 N2B27 중간 II(표 1)의10 mL에서 미적 세균 성 접시에 들어간다.</li>
		<li>2일째에, 2.3.2-2.3.3 단계에 설명된 바와 같이 세포 응집체를 수집하고 신선한 N2B27 배지 II의 10mL를 사용하여 배아 체를 재연한다.</li>
		<li>새로운 미동세균 접시에 이식하고 37°C에서 5%CO2 인큐베이터에서 2일 동안 분화를 허용한다. 현미경의 밑에 태아 바디의 대형을 확인하십시오.</li>
		<li>4일째되는 날, 배아시체를 수집합니다. N2B27 배지 II의 2mL를 가진 0.1% 젤라틴 코팅 6웰 플레이트에 잘 당 약 50개의 배아 체를 시드한다.</li>
		<li>모든 트랜스 RA 스톡 2 μL을 각 우물에 넣고 또 다른 4일 동안 분화를 유도합니다. 전체 배지(N2B27 배지 II를 1μM RA로)를 2일마다 교체합니다.</li>
	</ol></li>
	<li>프로토콜 7을 사용하여 단계 I 분화(표 1)<ol>
<li>약 2 x 104 mESC를 기초 분화 배지 의 2 mL 내에서 종자 0.1% 젤라틴 코팅 6 웰 플레이트에 잘. 플레이트를 37°C에서 5% CO2 인큐베이터에 넣고 6h에 부착할 수 있도록 합니다.</li>
		<li>PBS로 셀을 두 번 씻으시면 됩니다. 이어서, N2B27 배지 II의 2mL를 각 웰에 추가하고 5% CO2 인큐베이터에서 37°C에서 8일 동안 분화를 허용한다.</li>
		<li>2일마다 전체 N2B27 중간 II를 변경합니다.</li>
	</ol></li>
</ol>3. 세포 형태 관찰
<ol><li>반전 된 상 대비 빛 현미경에서 매일 위에서 언급 한 7 그룹의 분화 상태를 확인합니다.</li>
	<li>무작위로 최소 12개의 필드를 선택하고 사진을 찍어 D8에서 각 그룹의 형태학적 변화를 기록합니다.</li>
</ol>4. 면역 형광 염색
<ol><li>샘플 준비: 종자 mESC0.1% 젤라틴 코팅 커버립에 2단계에서 언급된 프로토콜을 사용하여 8일 동안 분화를 허용한다.</li>
	<li>린세:8일째에 인큐베이터에서 샘플을 꺼내 포부로 분화 매체를 제거합니다. 부드럽게 5 분 동안 PBS의 1 mL로 한 번 세포를 헹구십시오.</li>
	<li>고정: 각 샘플에 4% 파라포름알데히드 1mL을 추가하고 실온(RT)에서 20분 동안 셀을 수정합니다.</li>
	<li>헹구기: 고정 후, PBS 의 1 mL로 세포를 3번 부드럽게 헹구고, 각각 5분 동안 헹구십시오.</li>
	<li>침투성: PBS에 0.2% TritonX-100의 1mL을 각 샘플에 추가하고 RT에서 8분 동안 둡니다.</li>
	<li>헹구기: 투과화 후, PBS 3회 1mL로 세포를 부드럽게 헹구고 각각 5분간 헹구십시오.</li>
	<li>차단: PBS에 1mL의 염소 세럼을 각 샘플에 추가하고 RT에서 1h에 인큐베이션하여 비특이적 상호 작용을 차단합니다.</li>
	<li>1차 항체를 가진 배양<ol>
<li>PBS에서 5% 염소 세럼을 사용하여 1:100의 비율로 항 네스티닌 항체를 희석시.</li>
		<li>희석 된 항체의 500 μL을 다른 샘플에 적용하고 4 °C에서 하룻밤 동안 배양하십시오.</li>
	</ol>
</li>
	<li>헹구기: 항체를 제거하고 각각 8분 동안 PBS 3회 1mL로 시료를 부드럽게 헹구십시오.</li>
	<li>이차 항체를 가진 배양<ol>
<li>PBS에서 5% 염소 세럼을 사용하여 알렉사 플루어 488 라벨 염소 안티 마우스 IgG의 비율로 희석 1:500.</li>
		<li>희석 된 항체의 500 μL을 다른 샘플에 적용하고 RT에서 2 시간 동안 어둠 속에서 배양하십시오. 참고: 형광 이차 항체를 적용한 후, 형광 담금질을 방지하기 위해 어둠 속에서 모든 후속 단계를 수행합니다.</li>
	</ol>
</li>
	<li>헹구기: 이차 항체를 제거하고 각각 8분 동안 PBS 3회 1mL로 시료를 부드럽게 헹구십시오.</li>
	<li>핵 염색 및 장착<ol>
<li>DAPI 마운팅 배지 1방울을 깨끗한 마이크로슬라이드에 놓습니다.</li>
		<li>조심스럽게 플레이트에서 샘플을 꺼내 서 아래로 셀을 가진 DAPI 장착 매체위에 샘플을 배치. RT에서 5 분 동안 어둠 속에서 둡니다.</li>
		<li>흡수성 용지로 과도한 DAPI 마운팅 매체를 제거합니다.</li>
	</ol>
</li>
	<li>형광 현미경 검사기 관찰<ol>
<li>형광 현미경 검사의 밑에 표본을 놓고 적당한 필터를 사용하여 DAPI와 알렉사 플루올48에 대한 신호를 검출합니다.</li>
		<li>각 샘플에 대해 10-15개의 서로 다른 시각적 필드를 고르고 무작위로 선택하고 CCD 카메라로 이미지를 기록합니다.</li>
	</ol>
</li>
</ol>5. NPC에서 뉴런으로의 차별화 (단계 II)
<ol><li>가장 높은 차별화 효율을 가지는 단계 2.4에 상세한 프로토콜 3(8일, 표 1)을사용하여 단계 I 분화 하에서 mESC 유도체를 준비합니다(그림 3참조). 참고: 1상 제분화 후, 건강하고 고수율의 NPC를 보장하기 위해 위에서 언급한 세포 형태 관찰 및 면역형광 분석방법을 사용하여 품질 관리를 수행해야 합니다.</li>
	<li>약 5 x 105 mESC 유도체를 기초 분화 배지 의 2mL 내에서 0.1% 젤라틴 코팅 6웰 플레이트에 잘 뿌려. mESC 유도체를 단계 II 프로토콜 1, 프로토콜 2 및 프로토콜 3로 개별적으로 3그룹으로 임의로 분할합니다.</li>
	<li>플레이트를 37°C에서 5% CO2 인큐베이터에 넣고 6h에 부착할 수 있도록 합니다. PBS 2mL로 두 번 씻으시면 됩니다.</li>
	<li>기저 분화 매체 I, N2B27 배지 I 및 N2B27 배지II(표 2)의2mL을 각각 위의 그룹의 각 웰에 추가합니다.</li>
	<li>플레이트를 인큐베이터에 넣고 10일 동안 차별화할 수 있습니다. 해당 매체를 2일마다 변경합니다.</li>
	<li>분화 상태를 확인하고 3단계에서 언급한 대로 형태학적 변화를 기록합니다.</li>
	<li>18일째에, 뉴런의 생성을 평가하고(β-Tubulin III 양성)을 평가하고 4단계에서 사용하는 3개의 프로토콜의 분화 효율을 결정한다.</li>
</ol>

<ol>
	<li>Vieira, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+differentiation+into+mature+neurons:+mechanisms+of+regulation+and+biotechnological+applications.">Neural stem cell differentiation into mature neurons: mechanisms of regulation and biotechnological applications.</a> Biotechnology Advances. 36 (7), 1946-1970 (2018).</li><li>Yang, J. R., Lin, Y. T., Liao, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+embryonic+stem+cells+on+Parkinson's+disease+therapy.">Application of embryonic stem cells on Parkinson's disease therapy.</a> Genomic Medicine, Biomarkers, and Health Sciences. 3 (1), 17-26 (2011).</li><li>Liu, C., Zhong, Y. W., Apostolou, A., Fang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+human+embryonic+stem+cells+as+an+in+vitro+tool+for+the+study+of+the+expression+patterns+of+the+neuronal+cytoskeleton+during+neurogenesis.">Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis.</a> Biochemical and Biophysical Research Communications. 439 (1), 154-159 (2013).</li><li>Khramtsova, E. A., Mezhevikina, L. M., Fesenko, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+mouse+embryonic+stem+cells+modified+by+the+Neural+Growth+Factor+(NGF)+gene.">Proliferation and differentiation of mouse embryonic stem cells modified by the Neural Growth Factor (NGF) gene.</a> Biology Bulletin. 45 (3), 219-225 (2018).</li><li>Yoshimatsu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+efficacy+of+small+molecules+for+neural+differentiation+of+common+marmoset+ESCs+and+iPSCs.">Evaluating the efficacy of small molecules for neural differentiation of common marmoset ESCs and iPSCs.</a> Neuroscience research. , (2019).</li><li>Kothapalli, C. R., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+matrix+microenvironment+for+targeted+differentiation+of+embryonic+stem+cells+into+neural+and+glial+lineages.">3D matrix microenvironment for targeted differentiation of embryonic stem cells into neural and glial lineages.</a> Biomaterials. 34, 5995-6007 (2013).</li><li>Gazina, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+derivation+and+differentiation+of+mouse+embryonic+stem+cells+generating+synchronous+neuronal+networks.">Method of derivation and differentiation of mouse embryonic stem cells generating synchronous neuronal networks.</a> Journal of Neuroscience Methods. 293, 53-58 (2018).</li><li>Ohnuki, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hanging+drop+culture+conditions+on+embryoid+body+formation+and+neuronal+cell+differentiation+using+mouse+embryonic+stem+cells:+Optimization+of+culture+conditions+for+the+formation+of+well-controlled+embryoid+bodies.">Effects of hanging drop culture conditions on embryoid body formation and neuronal cell differentiation using mouse embryonic stem cells: Optimization of culture conditions for the formation of well-controlled embryoid bodies.</a> Journal of Bioscience and Bioengineering. 115 (5), 571-574 (2013).</li><li>Wongpaiboonwattana, W., Stavridis, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+mouse+embryonic+stem+cells+in+serum-free+monolayer+culture.">Neural differentiation of mouse embryonic stem cells in serum-free monolayer culture.</a> Journal of Visualized experiments. (99), e52823 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+neuronal+differentiation+of+embryonic+stem+cells+in+vitro.">An optimized method for neuronal differentiation of embryonic stem cells in vitro.</a> Journal of Neuroscience Methods. 330 (15), 108486 (2020).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+optimal+suspension+culture+time+for+the+osteoblastic+differentiation+of+human+induced+pluripotent+stem+cells+using+the+embryoid+body+method.">Investigation of the optimal suspension culture time for the osteoblastic differentiation of human induced pluripotent stem cells using the embryoid body method.</a> Biochemical and Biophysical Research Communications. 515 (4), 586-592 (2019).</li><li>Lu, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-trans+retinoic+acid+promotes+neural+lineage+entry+by+pluripotent+embryonic+stem+cells+via+multiple+pathways.">All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways.</a> BMC Cell Biology. 10, 57 (2009).</li><li>Kanungo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+in+P19+stem+cell+differentiation.">Retinoic acid signaling in P19 stem cell differentiation.</a> Anticancer Agents in Medicinal Chemistry. 17 (9), 1184-1198 (2017).</li><li>Rochette-Egly, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+and+mouse+embryonic+stem+cell+differentiation:+Cross+talk+between+genomic+and+non-genomic+effects+of+RA.">Retinoic acid signaling and mouse embryonic stem cell differentiation: Cross talk between genomic and non-genomic effects of RA.</a> Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1851 (1), 66-75 (2015).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+maintains+self-renewal+of+murine+embryonic+stem+cells+via+a+feedback+mechanism.">Retinoic acid maintains self-renewal of murine embryonic stem cells via a feedback mechanism.</a> Differentiation. 76 (9), 931-945 (2008).</li><li>Wu, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid-induced+upregulation+of+miR-219+promotes+the+differentiation+of+embryonic+stem+cells+into+neural+cells.">Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells.</a> Cell Death Disease. 8, 2953 (2017).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+regulates+germ+cell+differentiation+in+mouse+embryonic+stem+cells+through+a+Smad-dependent+pathway.">Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway.</a> Biochemical and Biophysical Research Communications. 418 (3), 571-577 (2012).</li><li>Nakayama, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+neuronal+induction+of+the+P19+embryonic+carcinoma+cell+line.">A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line.</a> Journal of Neuroscience Methods. 227, 100-106 (2014).</li><li>Bibel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+into+a+defined+neuronal+lineage.">Differentiation of mouse embryonic stem cells into a defined neuronal lineage.</a> Nature neuroscience. 7 (9), 1003-1009 (2004).</li><li>Coyle, D. E., Li, J., Baccei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Differentiation+of+Retinoic+Acid-Induced+Human+Pluripotent+Embryonic+Carcinoma+Stem+Cell+Neurons.">Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons.</a> PLoS ONE. 6 (1), 16174 (2011).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Developmental Biology. 275, 124-142 (2004).</li></ol>

저자는 공개 할 것이 없습니다.

본 연구에서는, 우리는 mESC에서 신경 분화를 위한 간단하고 효과적인 방법을 설치했습니다, 저렴한 비용과 쉽게 얻을 수 있는 물질. 이 방법에서, RA 유도6일 뒤에 배아체 형성의 2일은 효과적으로 NPC로 mESC의 분화를 촉진할 수 있다 (단계 I-프로토콜 3). 상 II 분화의 경우, N2B27 배지 II(Phase II-프로토콜 3)는 NPC로부터 뉴런으로의 분화를 가장 효과적으로 유도한다. 성공을 보장하기 위해 몇 가지 중요한...

배아 줄기 세포 (ESC)는 다능하며 신경 전구체 세포 (NPC)로 분화하고 특정 조건1하에서뉴런으로 분화 할 수 있습니다. ESC 기반 신경 발생은 신경 발생을 모방하는 최고의 플랫폼을 제공하므로 발달 생물학 연구를위한 유용한 도구로 봉사하고 잠재적으로 재생 의학2,3에도움이됩니다. 지난 수십 년 동안, 형질전환법(4)과 같은 배아 신경발생을 유도하기 위한 많은 전략이 보고되었으며, 소분자5를이용하여, 3D 매트릭스 마이크로환경6,공동배양기술7을이용하여 보고되었다. 그러나 이러한 프로토콜의 대부분은 조건이 제한적이거나 작동하기 어렵기 때문에 대부분의 실험실에서 의사용에 적합하지 않습니다.
mESC로부터 효율적인 신경 분화를 달성하기 위한 조작이 용이하고 저렴한 방법을 찾기 위해, 여기에 조합 선별 전략이 사용되었습니다. 도 1에설명된 바와 같이, 배아 신경 발생의 전체 과정은 2단계로 나뉘었다. 1단계는 MESC에서 NPC로의 분화 과정을 말하며, 위상 II는 NPC에서 뉴런으로의 후속 분화와 관련이 있습니다. 간편한 작동, 저렴한 비용, 용이한 재료 및 높은 분화 효율의 원리에 기초하여, 단계 I의 7개의 프로토콜과 단계 II의 3개의 프로토콜은 전통적인 부착단 단층 배양 시스템 또는 배아 체형성 시스템8,9에기초하여 선택되었다. 두 단계에서 프로토콜의 분화 효율은 세포 형태 관찰 및 면역 형광 분석을 사용하여 평가되었다. 각 단계에서 가장 효율적인 프로토콜을 결합하여 mESC에서 신경 분화를 위한 최적화된 방법을 수립했습니다.

<table>
	<tbody>
		<tr>
			<td>Anti-Nestin antibody [Rat-401]</td>
			<td>Abcam</td>
			<td>Ab11306</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III antibody produced in rabbit</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -80 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-Labeled Goat Anti-Mouse IgG</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>stored at -20 &#176;C, and protect from light</td>
		</tr>
		<tr>
			<td>CHIR-99021 (CT99021)</td>
			<td>Selleck</td>
			<td>S1263</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-Labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0516</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>DME/F-12 1:1 (1x)</td>
			<td>HyClone</td>
			<td>SH30023.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Gibco</td>
			<td>CM0635B</td>
			<td>stored at room temperature</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Immunol Staining Primary Antibody dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor human</td>
			<td>Sigma</td>
			<td>L5283</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI - Aqueous, Fluoroshield</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson</td>
			<td>005-000-121</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nonadhesive bacterial dish</td>
			<td>Corning</td>
			<td>3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin Solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PD0325901(Mirdametinib)</td>
			<td>Selleck</td>
			<td>S1036</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>stored at -80 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Strain 129 Mouse Embryonic Stem Cells</td>
			<td>Cyagen</td>
			<td>MUAES-01001</td>
			<td>Maintained in feeder-free culture system</td>
		</tr>
		<tr>
			<td>Stem-Cellbanker (DMSO free)</td>
			<td>ZENOAQ</td>
			<td>stem cellbanker DMSO free</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1X) Solution</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>6 - well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>NUNC</td>
			<td>339650</td>
			<td></td>
		</tr>
	</tbody>
</table>

neuronal differentiation from mouse embryonic stem cells in vitro

마우스 배아 줄기 세포 (mESC)의 신경 분화는 신경 발생에 관련 되 고 잠재적으로 재생 의학에 도움이 되는 주요 메커니즘을 해명 하기 위한 잠재적인 도구. 여기서 는 편생스크리닝 전략을사용하여 체외에서 mESC로부터 뉴런 분화를 위한 효율적이고 저렴한 비용 방법을 확립했습니다. 여기에 정의된 조건하에서, 2일 배아 체형성 + 6일 망막산 유도 프로토콜은 잘 쌓인 및 중성염과 같은 A2lox 및 129개의 유도체의 형성에 의해 볼 수 있듯이 mESC에서 신경 전구체 세포(NPC)로 빠르고 효율적인 분화를 허용합니다. 배아 체의 건강한 상태와 레티노산(RA)이 적용되는 시점뿐만 아니라 RA 농도는 이 과정에서 매우 중요합니다. NPC에서 뉴런으로 의 후속 분화에서, N2B27 중간 II (신경 물질 매체에 의해 보충) 더 나은 장기 유지 보수 및 신경 세포의 성숙을 지원할 수 있습니다. 제시된 방법은 매우 효율적이고, 비용이 낮으며, 작동이 용이하며, 신경생물학 및 발달 생물학 연구를 위한 강력한 도구가 될 수 있습니다.

2일 배아체 형성 + 6일 RA 유도는 MESC를 NPC로 분화시키는 데 가장 적합합니다(1상). MESC를 NPC(Phase I)로 균등화하는 최적의 프로토콜을 결정하기 위해, 7개의 프로토콜은 A2lox 및 129mESC(표1)에서모두 테스트되었으며 각 그룹의 분화 상태는 광 현미경을 사용하여 모니터링하였다. 도 3A에도시된 바와 같이, 대부분의 A2lox 및 129 유도체는 "2일 배아 체형성 + 6일 RA 유도" 치?...

Watch this Scientific Journal Video about Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro at JoVE.com

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

여기서, 우리는 배아 줄기 세포에서 뉴런으로 빠르고 효율적인 분화를 지시하는 저렴한 비용과 조작하기 쉬운 방법을 설립했습니다. 이 방법은 실험실 간의 대중화에 적합하며 신경 학적 연구를위한 유용한 도구가 될 수 있습니다.

시험관 내 마우스 배아 줄기 세포로부터의 신경 분화

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

neuronal-differentiation-from-mouse-embryonic-stem-cells-in-vitro

Research

JoVE Journal

Neuroscience

8.8K 조회수.  Wuhan Center for Disease Control and Prevention. 여기서, 우리는 배아 줄기 세포에서 뉴런으로 빠르고 효율적인 분화를 지시하는 저렴한 비용과 조작하기 쉬운 방법을 설립했습니다. 이 방법은 실험실 간의 대중화에 적합하며 신경 학적 연구를위한 유용한 도구가 될 수 있습니다.

비디오: Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포에서 인간의 연결을 Progenitors과 뉴런의 효율 유도

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

연구

신경과학

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

배아 복부 Midbrain의 Organotypic 슬라이스 문화 : Dopaminergic의 연결을 개발 연구하는 시스템 체외에서</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

배아 Murine 신경 튜브에서 신경 크레스트 세포의 분리 및 문화

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

인간의 다 능성 줄기 세포에서 도파민 신경 세포의 분화를 감독

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

대뇌 피 질의 Interneuron 선구자로 마우스 배아 줄기 세포의 분화

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

절연 및 마우스 배아 신경 줄기 세포의 문화

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

생체 내 신경 회로 연결성 모방을 위한 체외 쐐기 슬라이스 준비

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

섬유아세포에서 신경 전구세포로의 직접 변환을 이용한 신경질환 시험관 모델링 및 성상세포로의 분화

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

생체 내 마우스 열등올리브의 칼슘 이미징

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

마우스 성상세포의 분리 및 직접 뉴런 리프로그래밍

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...